Natural killer (NK) cells are cytolytic lymphoid cells of the innate immune system which identify cells with dysregulated MHC class I expression. The lack of normal MHC class I expression, often a consequence of tumorigenesis or viral infection, is detected by MHC class I allele-specific receptors on NK cells. Potential targets are protected from lysis if they express sufficient levels of the appropriate class I molecule. In order to advance our understanding of MHC recognition by NK receptors, we have determined the crystal structure, at 2.3 Angstrom units resolution, of the mouse lectin-like NK receptor Ly49A bound to its class I ligand, H-2Dd. The structure of the complex reveals a two-site mode of binding between dimeric Ly49A and H-2Dd. At one interface, an Ly49A subunit contacts H-2Dd at a site away from the peptide-binding groove which includes the N-terminal residues of the alpha1 helix and C-terminal residues of the alpha2 helix. This site represents the known trans interaction of Ly49A with H-2Dd. The structure also shows a second interface, which overlaps the CD8-binding site and is compatible with a cis interaction between Ly49A and H-2Dd on the same NK cell. This work provides a starting point for further structural studies of NK receptors. Our aim is to elucidate the molecular basis for ligand recognition by three key receptors known to regulate NK cell-mediated cytolysis: 1. Determination of the crystal structures of additional members of the Ly49 family (Ly49C, G2 and I) complexed with MHC class I in order to understand the specificity of different LY49 receptors for different class I alleles. We will also seek to precisely define the role carbohydrate in Ly49/MHC recognition by comparing the affinity of Ly49A for glycosylated and unglycosylated H-2Dd and by determining the structure of Ly49A complexed with the glycosylated class I protein. 2. Determination of the crystal structure of mouse 2B4, a member of the immunoglobulin superfamily that is expressed on all NK cells and on T cells that exhibit non-MHC-restricted (NK-like) lysis. The ligand for 2B4 has recently been identified as CD48 and the 2B4/CD48 interaction has been implicated in the regulation of NK cell function. 3. Determination of the structural basis for MHC recognition by the human NK inhibitory receptor ILT2, which interacts with the MHC class Ib molecule HLA-G. To this end, we have expressed a soluble form of ILT2 in Drosophila cells for use in direct binding and co-crystallization experiments with HLA-G.